RESEARCH ARTICLE | MARCH 08 2024

Evaluation serum level of IL-17, IL-23 and IL-35 in patients

with bacterial prostatitis in Najaf Province
Kais Khudhair AL Hadrawi  ; Mayyada F. Darweesh

AIP Conf. Proc. 3092, 020008 (2024)

https://doi.org/10.1063/5.0200466

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11 March 2024 14:06:44

 Evaluation Serum Level of IL-17, IL-23 and IL-35 in
Patients with Bacterial Prostatitis in Najaf Province

Kais Khudhair AL hadrawi1,a) and Mayyada F. Darweesh2,b)

1
Radiology Techniques Department, College of Medical Technology, Islamic University, Najaf, Iraq.
2
Biology, Faculty of Science, University of Kufa, Najaf, Iraq
a)
Corresponding author: kaisalhadrawii@gmail.com
b)
mayada.alsagheeri@uokufa.edu.iq

Abstract. This investigation aims to evaluate the levels of IL-17, IL-23, and IL-35 in prostate inflammation patients and
identify bacterial agents to gain further insights into outcome of prostatitis and improve treatment options for this
condition. A patient - healthy study was design on 90 prostatitis patients and 30 healthy control subjects. Blood and
semen samples were collected from all participant to measure concentration of interlukine-17, 23, and 35 by ELISA
assay, identify bacterial agents by culturing on general and differential media and confirmed by Vitek system, also
evaluate semen characteristics.
The study found that E. coli, K. pneumonia, and S. aureus were the leading causes of prostatitis infection. The IL-17
and IL-23 concentration were highly elevated in both acute and chronic prostatitis patients, with the highest levels
observed in patients with chronic prostatitis. A decrease in IL-35 levels was also observed in both acute and chronic

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prostatitis. The study found IL-17 and IL-23 have a positive association, while IL-17 and IL-35 have a negative
correlation.

Keywords. IL - 23 , IL - 17 , IL-35 , bacteria ,infertility , prostatitis

INTRODUCTION
Prostatitis is an infection that can result in bacterial inflammation of the prostate gland. It is a common condition,
affecting 10% of the male population worldwide, prostatitis is thought to affect 50% of men at some point in their
lives. Symptoms of prostatitis include pain or discomfort in the lower pelvic area, difficulty urinating, and frequent
urination[1]. Acute and chronic inflammatory infiltrates can both be found in the prostate, they may occur silently
or come with prominent symptoms [2]. Acute or chronic inflammation in the prostate can lead to infertility due to
several mechanisms, involving dysregulated spermatogenesis, blocked sperm transport, and reduced accessory
gland function. During the inflammatory phase, these mechanisms may result in lower semen quality, which may
have a detrimental effect on fertility. These mechanisms can lead to decreased semen quality during the
inflammatory phase which can negatively impact fertility [3].
Cytokines are multifunctional glycoproteins released by several male genital tract tissues and implicated in a
variety of activities that may have an impact on steroidogenesis, spermatogenesis, sperm functions, and fertility
regulation steroidogenesis, spermatogenesis, sperm functions, and fertility regulation [4].
Inflammation and infection of the testis and male genital tracts are linked to IL-17, which may play a significant
role in the pathophysiology of male infertility [5]. Th17 cells are involved in the immune response during prostate
infections, these cells secrete cytokines such as interleukin IL-17A IL-26, IFN-γ, CCL20 and IL-22 that associated
with inflammation, tissue damage, and scarring, which may lead to long-term complications such as infertility [6].
Pro-inflammatory cytokine IL-23 is thought to be a major participant in the etiology of many inflammatory and
autoimmune illnesses, including prostatitis. It also plays a function in immunological response to infection and
inflammation [7].
IL-35 is anti-inflammatory cytokine that plays an important role in regulating the immune response, it can inhibit
the secretion of pro-inflammatory cytokines such as interferon -γ and IL-12, while upregulating the expression of
IL-10. This helps to reduce inflammation, tissue damage and promote proliferation of T-reg cells and B-reg cells.
This helps to balance the immune response and prevent an overactive inflammatory response [8]. Therefore, this
study design to evaluate role of these cytokines on prostatitis disease outcome.

2nd International Conference on Engineering and Science to Achieve the Sustainable Development Goals
AIP Conf. Proc. 3092, 020008-1–020008-8; https://doi.org/10.1063/5.0200466
Published by AIP Publishing. 978-0-7354-4884-1/$30.00

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 METHODS AND SUBJECTS
Ninety patients who attended Infertility and Sterility Center in AL-Saddar Medical City / Al-Najaf province
between February and July of 2021 were included in a hospital-based case-control study. In addition to thirty
healthy males as control group were included. ELISA system's used to evaluation IL-23, IL-17, and IL-35 levels
(Elabscience®, China), Each participated had approximately 3 ml of blood drawn, separated into gel tubes, and
stored at -20 ºC until needed. To identify bacterial infection, semen specimen collected from patients and a loop of
semen was cultured on enrichment and differential media incubated at 37 oC for 24 h then positive bacterial growth
were subculture and single colony identify by Vitek system. The density, motility, viability, morphology, volume,
appearance, and semen liquefaction of the semen were all measured according to the instructions of the
(WHO,2010).
Inclusion Criteria: Acute and ongoing prostatitis patients were included. Individuals who were on ongoing
antibiotic therapy, those with chronic epididymitis/orchitis, prostate cancer, or benign prostate syndrome were
excluded from the study.
The study was authorized by the institutional ethics committees of AL-Saddar Hospital and the College of
Science at Kufa University. Also, each participant provided written, informed consent before to participating in the
study. This research was carried out in accordance with the Declaration of Helsinki, which is the World Medical
Association's code of ethics for studies involving humans.

STATISTIC EVALUATION
Using Scheffe's approach, the mean ± standard deviation were employed in the univariate ANOVA test. P≤0.05
was the statistical cutoff for comparing patients to healthy controls.

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RESULTS
This study included 90 prostatitis patients which classified as : acute prostatitis 32 (35.5%) and chronic
prostatitis 58 (64.5%) patients . The age disparity of the patients explain that 31-40 years had the highest frequency
of prostatitis, as shown in the table (1).
TABLE 1. Divided prostatitis patients into infection types and age.

Prostatitis Healthy P
Age properties
n=90 n=30
Age range 20-56 19-50 0.301 †
NS
Age mean 44.36 40.96

Standard deviation 12.50 13.43

Age groups
< 25, n (%) 0.292 ¥ NS
16(18.3) 4(13.3)

25-45 ,n (%) 42 (48.3) 17(56.7)

≥ 45, n (%) 32(33.4) 9(30)

Type of infection

Acute 32(35.5%) Nil

chronic 58(64.5%) Nil

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 NS: No significant, n: number, P: probability

The study results showed that out of 90 semen specimens, E. coli represent 30 (33.2%) isolates, S.aureus were 21
(24.6%) isolates , 15 (15.4%) isolates belonged to K. pneumonia, and 9 (10.2%), 8 (8.8%), and 7 (7.8%) isolates
were Enterobacter spp., P. aerogenosa, and P. mirabilis, respectively.
TABLE 2. bacterial agents associated with prostatitis disease

Bacterial isolates Colony morphology Total organism

N(%)
G-ve
E.coli Small, circular, slightly raised, smooth 30 (33.2)
K.pneumonia Circular, mucoidconvex, small colonies, 21(24.6)
capsulated
Enterobacter spp. smooth, irregularly round colonies 9 (10.2%)
P. aeruginosa Small, rough colony, flat edges 8(8.8%)
P. mirabilis translocate rapidly across the surface of 7(7.8%)
agar plates, resulting “swarming” motility.
G +ve
S .aureus Circular, pinheaded colonies, convex with 15 (15.4%)
entire margins
G+ve: gram positive bacteria, G-ve: gr negative bacteria

IL-17 Concentration in Patients and Healthy

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The concentration of IL-17 revealed a significant increase (P< 0.05) in all prostatitis patients' group (142.1±
23.01) pg/ml than in the healthy control group (18.2 ± 5.8). Also, within prostatitis patients, chronic cases higher
than acute cases (87.6 ±20.7 ;54.7 ± 11.3) pg /ml respectively with significant different (P value < 0.05) compare to
controls Figure (1).

160
IL-17 Pg/ml 142.1± 23.01
140

120

100 87.6 ±20.7

80
54.7 ± 11.3
60

40

20 18.2 ± 5.8
0
Acute chronic all patient healthy

FIGURE 1. Serum level IL-17 in acute , chronic prostatitis and healthy.

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 Determine the Level of IL-23 in the Serum of Prostatitis and Control

The results appear that level of IL-23 in prostatitis was 188.9±24.7 pg/ml and according to acute and chronic
infection were 104.8±13.5 and 84± 29.4 pg /ml respectively compared to the healthy group 15.3 ± 2.5 with
significant differences at p≤ 0,05 , figure ( 2).

188.9±24.7 IL-23 Pg/ml

200
180
160
140
120 104.8±13.5

100 84± 29.4

80
60
40 [VALUE]± 2.5
20
0
all patients Acute chronic healthy

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FIGURE 2. Serum level IL-23 in acute , chronic prostatitis and healthy.

IL-35 Levels Based on the Kind of Prostatitis

The result confirmed that IL-35 concentration in prostatitis (18.4 ±3.6) pg/ml compare to healthy men (48.5±
13.75). The mean concentration of IL-35 in acute and chronic prostatitis decrease to (22.4±5.4 ;12.6±3.9) compare
to control group. Figures ( 3 ).

48.5 ± 13.7

50 P value = 0.035 **

40

30 22.4 ± 5.4
18.4 ±3.6
IL-35

12.6 ± 3.9
20

10

0
healthy acute chronic all patients

FIGURE 3. IL-35 serum levels in chronic, acute prostatitis and healthy mean

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 Comparison between IL-17 and IL-23

This search found a positive relation between the of IL-17 and IL-23 in patients with prostatitis. This means
that as the level of IL-17 increases, the level of IL-23 also increases, as shown in Figure ( 4 ).

200
y = 0.1004x + 90.038
180
R² = 0.0133
160 r= 0.1153
140
120
IL-23

100
80
60
40
20
0
0 50 100 150 200 250 300
IL-17

FIGURE 4. Correlation between IL-17 and IL-23 in prostatitis patients.

Correlation between IL-17 and IL-35

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A positive correlation with significant differences between cytokine-17 with cytokine-35 in prostatitis, as
shown in Figure (5) .

25
y = 0.0056x + 13.7
R² = 0.0014
20 r= 0.0374

15
IL-35

10

0
0 50 100 150 200 250 300
IL-17

FIGURE 5. Correlation between IL-17 and IL-35 in prostatitis patients.

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 Correlation between IL-23 and IL-35

The search found a positive correlation with significant differences between IL-23 with IL-35 ,as shown in
Figure (6) .

25

20
y = 0.0181x + 12.45
15 R² = 0.0108
IL-35

r= 0.103
10

0
0 50 100 150 200
IL-23

FIGURE 6. Correlation between IL-23 and IL-35 in prostatitis patients.

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DISCUSSION

Patients Distribution Based on Ages and Kind of Infection

A study by [9] explain a 42-year-old mean age for patients with chronic prostatitis, with a range of 30-55 which
is consistent with this study. [10] indicates that the chronic prostatitis have a significant influence on a man's
quality of life. Also, [11]who observed that chronic prostatitis recorded high frequency compared to acute cases.
Similarly, [12] they confirmed that sperm DNA damage and abnormalities caused by chronic and acute prostatitis
increased with age by more than 40 years and had an adverse effect on fertility.
The present study found that E.coli , K.peumonia and S.aureus the main cause of infection and this results similar
to previous study by [13] whom found that E.coli and S.aureus most common bacterial causes in Iraqi prostatitis
patients . Also, [14] confirmed that E. coli is the main cause of chronic bacterial prostatitis (CBP), a type of
prostatitis caused by bacterial infection. The study also found that gram-positive bacteria, specifically E. faecalis, is
also a common causal agent of CBP. This suggests that these types of bacteria are commonly responsible for
causing chronic prostatitis as a results of pathogen's flexibility that afforded by an extensive number of virulence
factors , the ability to form biofilms, their ability to acquire the exchange of genetic material with other bacterial as
well as antimicrobial ability which has high rates of resistance to diverse antibiotic classes that generaly appear as
Multidrug resistance and extensively drug resistant (XDR) strains lead to persistence of long-term infections [15].
.

Concentration of IL-17 and IL-23 and Correlation between Them

This study in line with previous research that has found an association between increased levels of IL-17 and
prostatitis, particularly in chronic cases. In Iraq a study by [16] reported similar findings of increased IL-17 levels in
prostatitis patients. Research by [17] discovered that a rise in the levels of IL-17 can cause persistent inflammation
and infection. [18] reported that not only IL-17, but also INF-γ, and CXCL- 8 were raised in both acute and
persistent prostatitis compared to control men.

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 This study supports the findings of previous research that has identified a connection between chronic
prostatitis and elevated levels of IL-17, IL-1, and IL-23. [9] revealed that elevated levels of these cytokines which
are linked to persistent inflammation in the male genital tract, are present in patients with chronic prostatitis
.Similarly, a local study by [19] found a significant increase IL-17 concentration in infertile patients with prostatitis
compared to fertile patients and this suggests that prostatitis, particularly chronic cases, has an impact on fertility.
This study demonstrated that positive correlation between IL-17 and IL-23 are increased in indicating the
presence of inflammation in prostates patients compare to healthy group. This result in line with hypothesis of [20]
that found IL-17 connected to the pathophysiology of inflammatory and infection , Also [21] suggested the
involvement of the IL-23/Th17 pathway in the pathogenesis of blood transfusion infection . [22 ]they found that
IL-23 contributes significantly to the immune system's response against bacterial and fungal infections by driving
the differentiation and activation of Th17A and TNF-α , but its overproduction has been linked to many chronic
inflammatory diseases.

Concentration of Cytokine -35 in Prostatitis with Correlation between IL-17, IL-35

The result observed decreased level of IL-35 in prostatitis this in line with [19] who confirmed that infertile
prostatitis males and prostatitis patients had lower levels of IL-35 than infertile men. This results in consistence with
[23] observed that decreased serum levels of anti-inflammatory cytokine IL-4, IL-10 and IL-35 lead to a persistent
increased level of pro-inflammatory cytokines that causes chronic prostatitis in patients suffering from acute
prostatitis.
The results were agreement with the results of the study by [24] found that concentration of cytokines -17
and IL-35 in the peripheral blood of persistence prostatitis have opposite tendencies , they confirmed that IL-17
increases while the level of IL-35 decreases and these cytokines can serve as biomarkers for chronic inflammation

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of the urogenital tract and prostate infection in men. These findings suggest that the balance between these two
cytokines effect on the development or progression of prostatitis.
[25] observed that IL-35 stimulate the proliferation of Treg cells that inhibite the immune response and suppress
the differentiation of Th17 cells and prevent excessive tissue damage caused by the clearing of pathogens. The
study's suggests decreased levels of IL-35 may lead to the persistent inflammation as observed in prostatitis.

CONCLUSION

The imbalance between interleukins lead to persistence of prostatitis infection and the role of cytokines
dependent on time and level of each one so highly increased in decre level of pro-inflammatory cytokine IL-17
and IL-23 for long time causes decreased in anti-inflammatory IL35 and persistence infection

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